Over 2 million patients worldwide are treated annually with bone grafts to fill critical-sized craniofacial defects. Since autografts, the current gold standard treatment, introduce a high risk of donor site morbidity and have significant geometric constraints, tissue-engineered bone grafts (TEBGs) present a promising alternative with the potential to effectively regenerate geometrically complex, vascularized craniofacial tissues. However, translational application of TEBGs has been limited by significant knowledge gaps regarding the relationship between vascular structure and bone regeneration. The overall objective of this study is to develop and implement novel quantitative 3D imaging techniques to gain a fundamental understanding of how neo- vasculature impacts bone formation in TEBGs. While strategies have been developed to promote angiogenesis in regenerating bone, published reports demonstrate that the amount of vasculature formed within TEBGs has no correlation with the quantity or quality of regenerated bone. In native bone, microenvironmental interactions between vessels and bone cells are essential to bone growth and maintenance. In particular, scientists have recently identified a vessel phenotype high in CD31 and endomucin expression, termed ?Type H?, that is intimately associated with osteoprogenitors, and necessary for bone homeostasis. To determine whether Type H vessels are related to regenerating bone in TEBGs, this proposed work will integrate whole-mount immunostaining with a novel optical clearing method and light-sheet microscopy to image entire TEBGs (>mm3 volume) in 3D at single-cell resolution. Combining these technologies will enable unprecedented 3D quantitative characterization of vessel phenotypes and vessel-bone cell relationships. Vessel and bone formation will be evaluated with previously investigated TEBGs used to treat 4-mm murine critical-sized defects. In Aim 1, protocols for whole-mount immunostaining, clearing, and light-sheet imaging native murine calvaria and implanted calvarial TEBGs will be developed to enable 3D quantitative characterization of vessel phenotypes and spatial relationships between vessels and bone cells. In Aim 2, this 3D quantitative imaging technique will be applied to determine whether specific vessel phenotypes are correlated with enhanced bone formation in TEBGs. First, vessel and bone formation will be compared in TEBGs known to yield two distinct levels of bone regeneration in order to determine whether Type H vessel development contributes to bone healing. Second, the effects of angiogenic and osteogenic growth factors on vascularized bone formation in TEBGs will be evaluated to further elucidate the relationship of vessel phenotypes to bone regeneration. These findings will enable the development of targeted strategies to promote vascularized bone regeneration. This research will have a substantial positive impact on developing improved treatments for patients with debilitating craniofacial injuries.